The long-term objective of this concept-driven research program is to test the hypothesis that LAG-3 plays an important physiological role in regulating the expansion and survival of effector T-cells, and thus helps to regulate T-cell homeostasis'. Specific Aim 1: How does LAG-3 increase the number of expanding T-cells following antigen stimulation and what is the role of MHC class II molecules in this process? T-cells from Lag-3-deficient and wild-type mice carrying an MHC class I- or class Il-restricted TCR transgene will be used to determine whether LAG-3 increases the number of expanding T-cells by increasing proliferation or reducing apoptosis. We will also determine the role of MHC class II molecules in mediating LAG-3 function, and decipher the relationship between the timing of MHC class II ligation and LAG-3-mediated effector T-cell expansion. Specific Aim 2: What effect does LAG-3 have on the expansion and survival of effector cells after viral infection in vivo? The expansion and survival of influenza A-specific T-cells in the lung and the mediastinal lymph nodes following a primary and secondary infection of Lag-3-/- and Lag-3+/+ mice will be determined using MHC:peptide tetrameric complexes. The influence of LAG-3 expression on T-cell memory will also be examined. Specific Aim 3: What motifs in the cytoplasmic tail of LAG-3 mediate signal transduction? A series of LAG-3 mutants, which possess substitutions and/or deletions in the cytoplasmic tail, will be expressed by retroviral transduction in T-cells from Lag-3-/- mice carrying either the TEa or OVA TCR transgenes. Their effect on effector cell expansion following antigen stimulation will be determined. It is an established principle that the appropriate control of T-cell function and regulation of T-cell homeostasis is imperative. However, little is know about how the number of resident and circulating T-cells is controlled despite continual antigenic stimulation. Our preliminary data suggest that LAG-3 may contribute to this important process. This project will define the physiological role of LAG-3 and how it exerts its function.